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Newhouse K, Smith W A, Starrett M A, Schaefer T J and Singh B K, Tolerance to imidazolinone herbicides in wheat. The S653N mutation is among the five most common single-point mutations in AHAS genes that result in tolerance to imidazolinone herbicides in plants (Tan, S., Evans, R. R., Dahmer, M. L., charurbate com Singh, B. K., and Shaner, D. L. (2005) Imidazolinone-tolerant crops: History, current status and future. Several AHAS genes encoding AHAS enzymes that are tolerant to imidazolinone herbicides have been discovered in plants as naturally occurring mutations and through the process of chemically-induced mutagenesis. Alternatively, plants that transiently express the ZFP of choice, or to which the ZFP has been administered in a delivery vehicle, can be used. Transgenic and non-transgenic plants are also used as a preferred embodiment for examining regulation of endogenous gene expression in vivo. Specific amino acid modifications to the AHAS structural gene sequence have been described that alter the resulting proteins sensitivity to various structural classes of herbicides without a negative penalty on plant performance. The target site recognized by the DNA-binding domain therefore can be any suitable site in the target gene that will allow activation or repression of gene expression, optionally linked to a regulatory domain.

The activity of a particular ZFP can be assessed using a variety of in vitro and in vivo assays, by measuring, e.g., protein or mRNA levels, product levels, enzyme activity, you xxx porn transcriptional activation or repression of a reporter gene, using, e.g., immunoassays (e.g., ELISA and immunohistochemical assays with antibodies), hybridization assays (e.g., RNase protection, northerns, in situ hybridization, oligonucleotide array studies), colorimetric assays, amplification assays, enzyme activity assays, phenotypic assays, and the like. Pat. Nos. 6,453,242 and 6,794,136), one or more of the individual zinc fingers of a multi-finger binding domain can bind to overlapping quadruplet subsites. Pat. Nos. 6,479,626 and 7,851,216. Accordingly, one or more subsites, in a target site for a zinc finger binding domain, can be separated from each other by 1, 2, 3, 4, 5 or more nucleotides. The ZFP can be recombinantly expressed in a cell, recombinantly expressed in cells transplanted into a plant, or recombinantly expressed in a transgenic plant, as well as administered as a protein to plant or cell using delivery vehicles described below.

The cell may be a cell that naturally produces Cas protein, or a cell that naturally produces Cas protein and is genetically engineered to produce the endogenous Cas protein at a higher expression level or to produce a Cas protein from an exogenously introduced nucleic acid, which nucleic acid encodes a Cas that is same or different from the endogenous Cas. For such targeted DNA cleavage, a DNA-binding domain (e.g., zinc finger protein or TALE) is engineered to bind a target site at or near the predetermined cleavage site, and a fusion protein comprising the engineered zinc finger binding domain and a cleavage domain is expressed in a cell. The disclosed methods and compositions include fusion proteins comprising a DNA-binding domain (e.g., ZFP, TALE, etc.) and a regulatory domain or cleavage (e.g., nuclease) domain (or a cleavage half-domain), in which the DNA-binding domain, by binding to a sequence in cellular chromatin in one or more plant genes, induces cleavage and targeted integration of one or more exogenous sequences (including transgenes) into the vicinity of the target sequence.

Non-limiting examples of suitable AHAS target sites are shown in Table 3 and Table 13. The AHAS (also known as AHAS/ALS) genes are present in all major plant species including but not limited to maize, soybean, cotton, Arabidopsis, rice, sunflower, wheat, barley, sugarbeet and Brassica. Alternatively, two fusion proteins, each comprising a DNA-binding domain and a cleavage half-domain, are expressed in a cell, and bind to target sites which are juxtaposed in such a way that a functional cleavage domain is reconstituted and DNA is cleaved in the vicinity of the target sites. Upon binding of the DNA-binding portion of the fusion protein to the target site, the DNA is cleaved near the target site by the cleavage domain. However binding of, for example, a 4-finger binding domain to a 12-nucleotide target site, a 5-finger binding domain to a 15-nucleotide target site or a 6-finger binding domain to an 18-nucleotide target site, is also possible. Pat. No. 6,453,242. It will be clear to those skilled in the art that simple visual inspection of a nucleotide sequence can also be used for selection of a target site.

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